Serum protein electrophoresis (SPE) is a technique that could be considered one of the most useful diagnostic aids available to the clinician. The effect of storage time and temperature on the total proteins and electrophoretic fractions (albumin, α1-, α2-, β1-, β2-, and γ-globulins) was assessed in 24 healthy horses. All samples, collected by jugular vein puncture, were centrifuged and divided into 4 aliquots. The 1st aliquot was analyzed within 3 h from collection (time 0), the 2nd was refrigerated at +4°C for 24 h, the 3rd was refrigerated at +4°C for 48 h, and the last was frozen at -20°C for 48 h. One-way repeated-measures analysis of variance (ANOVA) showed a significant effect (P < 0.05) of the different storage conditions on the concentrations of all the parameters studied and significant variations in the percentages of albumin, α1-globulins, α2-globulins, and γ-globulins. Compared with time 0 the total protein concentration increased significantly after 48 h at -20°C, the albumin percentage decreased after 48 h at -20°C, the α1-globulin percentage increased after 24 h at +4°C, the α2-globulin percentage increased after 48 h at +4°C and at -20°C, and the γ-globulin percentage increased after 48 h at -20°C. The results should help veterinary practitioners handle and store equine serum samples appropriately. Further investigations at different storage times and temperatures could be useful.